Sudden cardiac death (SCD) claims the lives of approximately 350,000 Americans each year. Emerging evidence indicates an important role for genetic predisposition to SCD; however, the molecular determinants have remained elusive. The overall goal of this proposal is to investigate new mechanisms that underlie SCD through the application of innovative technology and the novel use of new genetic models of long QT syndrome 2 (LQT2). This multi-pronged approach includes the investigation of hormone-treated prepubertal, ovariectomized LQT2 rabbits to explore new mechanistic paradigms that underlie the effects of sex hormones on cardiac arrhythmias using in vitro and in silico experimental approaches that will integrate novel molecular, cellular, tissue, and theoretical models. Because gender and the sympathetic nervous system plays a key role in triggering SCD in LQT2, the influence of sex hormones and the autonomic factors on SCD risk will be studied in detail. The proposal is composed of Four Specific Aims: Aim 1: To characterize of the sex hormone induced changes in cardiac repolarization (QT duration and cardiac refractory periods), incidence of spontaneous TdP and sudden cardiac death with the use of telemetric ECG monitoring and in vivo invasive electrophysiological studies; Aim 2: To analyze the action potential duration, dispersion of repolarization, conduction, triggered activity and the heterogeneities in electrical/mechanical restitution, and conduction block using optical mapping of action potential and calcium transients. Aim 3: To define (based on the optical studies) the molecular determinants of the gender differences in cardiac repolarization and excitation by analyzing the gender or sex hormone related differences in the expression of genes coding for the or subunits of the repolarizing potassium currents, calcium currents, and proteins that control Ca++ cycling, and the adrenergic receptors with the use of protein expression studies and quantitative real-time PCR, and cellular electrophysiological studies; and Aim 4: To perform a comparative analysis of action potential and calcium-handling properties in myocytes isolated from hormone-treated LQT2 rabbits under control conditions and during autonomic receptor stimulation, using patch-clamp and fluorescent-indicator techniques. b) To use computer modeling to examine how experimentally defined cellular/molecular alterations in these LQT2 models influence afterdepolarizations and arrhythmogenic substrate formation in simulated cardiac tissue. These studies will contribute to the understanding of the mechanisms that trigger and maintain arrhythmias in hormone-treated rabbits, and therefore lead to a better understanding of gender-related arrhythmias in long QT syndrome.